The invention relates to reagents and methods for expressing gene products in cardiac cells or precursors to cardiac cells in vitro and in vivo.
Adult mammalian cardiomyocytes do not de-differentiate or re-enter the cell cycle. After being placed into cell culture, neonatal cardiomyocytes soon lose their ability to proliferate. Several cell lines, including P19 teratocarcinoma cells, embryonic stem (ES) cells, AT-1, H9c2, QCE-6, or 10T1/2 cells, have some molecular characteristics of cardiomyocytes. These cells are very difficult to manipulate, however, or are lacking important characteristics of cardiomyocytes. Because of these reasons, cultured neonatal cardiomyocytes from rats or mice are often used in in vitro systems, even though these cells are difficult to transfect (usually less than 0.1% transfection rate) and require long preparation procedures.
Recently, it has been demonstrated that immortalized cardiac myogenic (CMG) cells can be differentiated from mouse bone marrow stromal cells. This is evidence of the generation of cardiomyocytes from a tissue of extra-cardiac origin.
The possibility of bone marrow being an in vivo source of circulating cardiomyocyte progenitors has been previously suggested. A distribution of transplanted bone marrow-derived cells in a dystrophic mouse heart has been observed. Although the molecular characteristics of these cells were not identified, their location in the heart tissue indicated these cells were cardiomyocytes. Taken together, it appears that bone marrow stromal cells are an extra-cardiac source of cardiomyocytes in vivo, and in vitro induction of beating cardiomyocytes from a heterogeneous population of bone marrow cells is possible by the introduction of inductive agents such as 5-azacytidine.
The molecular mechanisms which guide development of cardiac cells (and the heart in general) in vertebrates have been the subject of intense investigation (Fishman and Chien, Cell 91: 153-156, 1997; Olson and Srivastava, Science 272: 671-676, 1996). In most vertebrates, the heart tissue initially develops as a crescent shaped mesodermal structure located anteriorly and laterally. This precardiac mesoderm is brought ventrally and caudally, by folding of the embryo, to a form a single midline heart tube with the inflow region located most rostrally. This heart tube undergoes looping, bringing the inflow, ventricular, and outflow regions of the heart into the alignment seen in the mature heart. Later, chamber septation occurs, valves develop in the atrioventricular (AV) junction as well as in the outflow tract, and the outflow tract itself is divided into two great vessels. Additional refinements occur with the development of the coronary arteries and the cardiac conduction system.
Heart development is governed by complex signals including inductive and positional signals from adjacent structures, as well as signals from a number of transcription factors (Fishman and Chien, Cell 91: 153-156, 1997; Lyons, Curr. Opin. Genet. Dev. 6: 454-460, 1996; Mohun and Sparrow, Curr. Opin. Genet. Dev. 7: 628-633, 1997; Olson and Srivastava, Science 272: 671-676, 1996). Since transcriptional factors have the ability to activate multiple genes, they are generally considered important regulators of organ development. A number of cardiac transcription factors have been identified that have important influences on the early stages of specification and differentiation of the cardiac mesoderm (Tanaka et al., Dev. Genet. 22: 239-249, 1998). Csx/Nkx2.5 (Komuro and Izumo, Proc. Natl. Acad. Sci. USA 90: 8145-8149, 1993; Lints et al., Development 119: 419-431, 1993), MEF-2C (Edmondson et al., Development 120: 1251-1263, 1994), GATA4 (Heikinheimo et al., Dev. Biol. 164: 361-373, 1994; Kelley et al., Development 118: 817-827, 1993) and dHAND and eHAND are members of four different classes of transcriptional factors all expressed in the heart at early stages of development. Targeted disruption of any one of these genes yields severe cardiac and extracardiac phenotypes, and results in death of the embryo between E9.5 and E10.5 of development.
The mouse Csx/Nkx2.5 gene is first expressed in the cardiac progenitor cells at embryonic day 7.5 (E7.5), and during this stage is detected principally in the heart and tongue (and, to a lesser extent, in spleen, stomach, liver, and larynx). The extra-cardiac expression of Csx/Nkx2.5 is markedly reduced after birth, however, and in the adult, the expression is confined to the heart. Mice in which the Csx/Nkx.2-5 gene has been deleted have no functional heart, causing an embryonic lethality by E9.5-11.5.
Most tissue-specific gene expression is controlled by enhancer and repressor sequences at the transcriptional level. Generally, to confer tightly-regulated expression, enhancers adopt complex regulatory mechanisms that require the collaboration of multiple transcription factors. The binding sites for these transcription factors may be many kilobases (kb) from the gene promoter and dispersed relative to each other.
It is desirable to be able to express genes in a cardiac cell-specific manner. This would be useful, for example, for the targeted expression of genes encoding therapeutic proteins for the treatment of damaged heart tissue. Moreover, to maximize the utility of stem cell-derived cardiomyocytes, for example, in the treatment of damaged heart tissue in humans and other animals, it is desirable to be able to rapidly purify cardiac cells from a potentially heterogenous cell population.
Accordingly, there is a need for the development of reagents and methods for achieving cardiac cell-specific gene expression. The present invention provides these reagents and methods.
In a first aspect, the invention features a substantially purified nucleic acid molecule comprising an enhancer element having: (a) 100% identity to 40 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 1 or SEQ ID NO.: 3; (b) at least 91% identity to 50 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 2; (c) at least 97% identity to 60 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 1 or SEQ ID NO.: 3; or (d) at least 95% identity to 70 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 1 or SEQ ID NO.: 3.
In a second related aspect, the invention features a substantially purified nucleic acid molecule comprising a cardiac-specific enhancer element derived from a human, wherein the enhancer element has at least 60% identity to 50 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 1, SEQ ID NO.: 2, or SEQ ID NO.: 3. Preferably, the element has at least 70% identity to 50 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 1, SEQ ID NO.: 2, or SEQ ID NO.: 3. More preferably, the identity is at least 80%, and most preferably, the identity is at least 90%, when compared to 50 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 1, SEQ ID NO.: 2, or SEQ ID NO.: 3.
Preferably, when expressed in vivo, the enhancer element is active in all four cardiac chambers. The enhancer element of the first or second aspect may be naturally occurring, or it may be non-naturally occurring.
Preferably, the enhancer element of the first or second aspect includes a binding site selected from the group consisting of Mef2, dHAND, GATA, TGF-xcex2, CarG, E-box, and Csx/Nkx2.5 binding sites. More preferably, the enhancer element includes at least two binding sites selected from this group. The enhancer element preferably also includes an Sp-1 binding site.
In a third aspect, the invention also features a substantially purified non-naturally occurring nucleic acid molecule that includes at least three transcription factor binding sites selected from Mef2, dHAND, GATA, TGF-xcex2, CarG, E-box, and Csx/Nkx2.5 binding sites. More preferably, the nucleic acid molecule includes four transcription factor binding sites, and most preferably includes five transcription factor binding sites selected from the aforementioned group. Preferably, the nucleic acid molecule, when operably linked to a promoter, increases activity of the promoter by at least two-fold in a cardiac cell-specific manner.
In a fourth aspect, the invention features a substantially purified nucleic acid molecule comprising an enhancer element having: (a) 100% identity to 50 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 6; (b) at least 97% identity to 60 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 6; (c) at least 93% identity to 70 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 6; or (d) at least 90% identity to 100 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 6.
In a fifth aspect, the invention features a substantially purified nucleic acid molecule that includes a cardiac-specific enhancer element derived from a human, wherein the enhancer has at least 45% identity to 50 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 6. Preferably, the element has at least at least 50% identity to 50 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 6, more preferably, the element has at least 60% identity to 50 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 6, and most preferably, the element has at least 75% identity or even 90% identity to 50 contiguous nucleotides of the nucleic acid molecule shown in SEQ ID NO.: 6. The element may be naturally occurring or non-naturally occurring.
In a fifth aspect, the invention features a substantially purified nucleic acid molecule comprising 50 contiguous nucleotides that have a sequence that is that at least 90% identical to 50 contiguous nucleotides of the nucleic acid molecule of SEQ ID NO.: 4 or SEQ ID NO.: 5.
In a sixth aspect, the invention features a DNA vector that includes the nucleic acid molecule of the first, second, third, fourth, or fifth aspects. The DNA vector can also have a promoter operably linked to a gene of interest. The gene of interest is preferably a cardiogenic gene (e.g., a gene encoding BMP2, BMP4, GATA4, dHAND, eHAND, MEF2C, IRX4, SRF, or Csx/Nkx2.5), a reporter gene (e.g., a gene encoding GFP, xcex2-gal, alkaline phosphatase, chloramphenicol acetyl transferase, or luciferase), a gene encoding a selectable marker (e.g., a gene that provides resistance to neomycin, kanamycin, or hygromycin), or a gene encoding a therapeutic protein (e.g., a growth factor, a cytokine, an anti-apoptotic factor, a pro-apoptotic factor, or a protein that improves cardiac function or repair).
In a seventh aspect, the invention features a method for inducing a cell to become a cardiac cell. The method includes (a) introducing into the cell or ancestor thereof a DNA vector that includes (i) the nucleic acid of the first, second, third, fourth, or fifth aspect; (ii) a promoter; and (iii) a cardiogenic gene operably linked to the promoter; and (b) placing the cell under conditions that result in expression of the cardiogenic gene operably linked to the promoter. Preferably, expression of the cardiogenic gene further enhances expression of cardiogenic genes by binding to cardiac-specific enhancer elements.
In an eighth aspect, the invention feature a method for specifically expressing a gene in cardiac cells, said method comprising introducing into the cell or ancestor thereof a DNA vector that includes (i) the nucleic acid of the first, second, third, fourth, or fifth aspect; (ii) a promoter; and (iii) the gene operably linked to the promoter. Preferably, the nucleic acid allows expression of the gene in a cardiac cell and does not express said gene in at least one cell that is not a cardiac cell.
In a ninth aspect, the invention features a method for determining the efficacy of a method of inducing target cells to produce or become cardiac cells, the method including: (a) introducing into at least one target cell (or an ancestor of the target cell) a DNA vector that includes (i) the nucleic acid of the first, second, third, fourth, or fifth aspect; (ii) a promoter; and (iii) a reporter gene operably linked to the promoter; (b) performing a method for potentially inducing the target cells to produce or become cardiac cells; and (c) determining the number or percentage of cells that are reporter gene-positive, wherein a higher number or percentage indicates a higher efficacy of the method of inducing stem cells to produce or become cardiac cells. Preferably, the target cells are stem cells such as bone marrow stem cells or embryonic stem cells.
In a tenth aspect, the invention features a method for determining the efficacy of a method of inducing target cells to produce or become cardiac cells, the method including: (a) introducing into at least one target cell (or an ancestor of the target cell) a DNA vector that includes (i) the nucleic acid of the first, second, third, fourth, or fifth aspect; (ii) a promoter; and (iii) a gene, encoding a selectable marker, operably linked to the promoter; (b) performing a method for potentially inducing the target cells to produce or become cardiac cells;(c) performing a drug selection, wherein cells expressing said gene encoding the selectable marker are capable of surviving in the presence of the drug and cells not expressing the gene encoding the selectable marker are not capable of surviving in the presence of the drug; and (d) determining the survival of cells following drug selection, wherein a higher cell survival indicates a higher efficacy of the method of inducing stem cells to produce or become cardiac cells. In this method, step (b) can be performed before or after step (c). Preferably, the target cells are stem cells such as bone marrow stem cells or embryonic stem cells.
In an eleventh aspect, the invention features a method of identifying a cardiac cell, including (a) introducing into the cell (or an ancestor of the cell) a DNA vector that includes (i) the nucleic acid of claim the first, second, third, fourth, or fifth aspect; (ii) a promoter; and (iii) a reporter gene operably linked to the promoter, whereby the cell expresses the reporter gene if the cell is a cardiac cell; (b) allowing sufficient time for the reporter gene to be expressed in cardiac cells; and (c) identifying the cardiac cells by the presence of the reporter gene. Preferably, the method is performed in vitro.
In a twelfth related aspect, the invention features a method of substantially purifying a cardiac cell from a heterogeneous population of cells, including: (a) introducing into at least a subset of cells in the population (or ancestors of these cells) a DNA vector that includes (i) the nucleic acid of the first, second, third, fourth, or fifth aspect; (ii) a promoter; and (iii) a reporter gene operably linked to the promoter, whereby a cell expresses the reporter gene if the cell is a cardiac cell; (b) determining whether a cell in the heterogeneous population is expressing the reporter gene; and (c) if the cell is expressing the reporter gene, purifying the cell from the heterogeneous population.
In a thirteenth aspect, the invention features a method of expressing a gene encoding a therapeutic protein in a cardiac cell. The method includes introducing to the cell (or an ancestor of the cell) a DNA vector that includes (i) the nucleic acid of the first, second, third, fourth, or fifth aspect; (ii) a promoter; and (iii) a gene encoding a therapeutic protein operably linked to the promoter such that the gene is expressed in cardiac cells. The cell may be a cardiac cell, or it may be a cell that is capable of differentiating as a cardiac cell. The method may be performed in vivo or in vitro. If the method is performed in vitro, the cell (or a descendent of the cell) can be grafted into a patient.
In all of the foregoing aspects of the invention, an enhancer element is defined as a nucleic acid sequence that when present, (i) increases in a cardiac cell expression of a gene to which it is operably linked by at least 25%, (ii) allows for gene expression in the heart tube prior to looping, (iii) allows for gene expression in all four heart chambers; or (iv) increases cardiac expression 100% more than it increases extracardiac expression. Preferably, in (i), the expression is increased by at least 50%, more preferably by 100%, and most preferably by 200%.
As used herein, by xe2x80x9cnucleic acidxe2x80x9d is meant either DNA or RNA. A xe2x80x9cnucleic acid moleculexe2x80x9d may be a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Unless otherwise specified, the left hand direction of the sequence of a single-stranded nucleic acid molecule is the 5xe2x80x2 end, and the left hand direction of double-stranded nucleic molecule is referred to as the 5xe2x80x2 direction.
By xe2x80x9cpromoterxe2x80x9d is meant a region of nucleic acid, upstream from a translational start codon, which is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A xe2x80x9chuman promoterxe2x80x9d is a promoter capable of initiating transcription in a human cell, and may or may not be derived from a human cell. A xe2x80x9cCsx/Nkx2.5 promoterxe2x80x9d is one derived from the promoter region of a Csx/Nkx2.5 gene and that, when operably linked to a heterologous nucleic acid molecule, is capable of initiating transcription of that molecule (when present in a transcription medium capable of supporting transcription) in a cardiac cell.
By xe2x80x9cenhancer elementxe2x80x9d is meant a nucleic acid sequence that, when positioned proximate to a promoter and present in a transcription medium capable of supporting transcription, confers increased transcription activity relative to the transcription activity resulting from the promoter in the absence of the enhancer domain.
By xe2x80x9coperably linkedxe2x80x9d is meant that two or more nucleic acid molecules (e.g., a nucleic acid molecule to be transcribed, a promoter, and an enhancer element) are connected in such a way as to permit transcription of the nucleic acid molecule in a suitable transcription medium.
By xe2x80x9cderived fromxe2x80x9d is meant that a the nucleic acid molecule was either made or designed from a second nucleic acid molecule, the derivative retaining important functional features of the nucleic acid molecule from which it was made or designed.
By xe2x80x9cexpression constructxe2x80x9d is meant a nucleic acid molecule that is capable of directing transcription. An expression construct of the present invention includes, at the least, a cardiac-specific enhancer element and a promoter. Additional elements, such as a transcription termination signal, may also be included, as described herein.
By xe2x80x9cvectorxe2x80x9d or xe2x80x9cexpression vectorxe2x80x9d is meant an expression system, a nucleic acid-based vehicle, a nucleic acid molecule adapted for nucleic acid delivery, or an autonomous self-replicating circular DNA (e.g., a plasmid). When a vector is maintained in a host cell, the vector can either be stably replicated by the cells during mitosis as an autonomous structure, incorporated within the genome of the host cell, or maintained in the host cell""s nucleus or cytoplasm.
By xe2x80x9ccardiac cellxe2x80x9d is meant a differentiated cardiac cell (e.g., a cardiomyocyte) or a cell committed to producing or differentiating as a cardiac cell (e.g., a cardiomyoblast or a cardiomyogenic cell).
By xe2x80x9ccardiac-specific enhancer elementxe2x80x9d is meant an element, operably linked to a promoter, that directs gene expression in a cardiac cell and does not direct gene expression in all tissues or all cell types. Cardiac-specific enhancers of the present invention may be naturally occurring or non-naturally occurring. One skilled in the art will recognize that the synthesis of non-naturally occurring enhancers can be performed using standard oligonucleotide synthesis techniques.
By xe2x80x9cplasmidxe2x80x9d is meant an autonomous DNA molecule capable of replication in a cell, and includes both plasmids designed for expression and plasmids designed for nucleic acid replication.
By xe2x80x9cheterologousxe2x80x9d is meant that the nucleic acid molecule originates from a foreign source or, if from the same source, is modified from its original form. Thus, a xe2x80x9cheterologous promoterxe2x80x9d is a promoter not normally associated with the duplicated enhancer domain of the present invention. Similarly, a heterologous nucleic acid molecule that is modified from its original form or is from a source different from the source from which the promoter to which it is operably linked was derived.
By xe2x80x9csubstantially pure nucleic acidxe2x80x9d is meant nucleic acid that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the nucleic acid. The term therefore includes, for example, a recombinant nucleic acid which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic nucleic acid of a prokaryote or a eukaryote cell; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also include, a recombinant nucleic acid which is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of a nucleic acid molecule (for example, DNA) which is inserted by artifice into a cell either transiently or permanently, and becomes part of the organism if integrated into the genome or maintained extrachromosomally. Such a transgene may include a gene which is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenic cellxe2x80x9d is meant a cell containing a transgene. For example, a stem cell transformed with a vector containing the expression vector of the present invention operably linked to a heterologous nucleic acid molecule can be used to produce a population of cells having altered phenotypic characteristics.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof.